Astrocytes are critical regulators of neuronal function in the central nervous system (CNS). We have previously shown that astrocytes in the spinal dorsal horn (SDH) have increased intracellular Ca2+ levels following intraplantar injection of the noxious irritant, formalin. However, the underlying mechanisms remain unknown. We investigated these mechanisms by focusing on the role of descending noradrenergic (NAergic) signaling because our recent study revealed the essential role of the astrocytic Ca2+ responses evoked by intraplantar capsaicin. Using in vivo SDH imaging, we found that the Ca2+ level increase in SDH astrocytes induced by intraplantar formalin injection was suppressed by ablation of SDH-projecting locus coeruleus (LC)-NAergic neurons. Furthermore, the formalin-induced Ca2+ response was dramatically decreased by the loss of α1A-adrenaline receptors (ARs) in astrocytes located in the superficial laminae of the SDH. Moreover, similar inhibition was observed in mice pretreated intrathecally with an α1A-AR-specific antagonist. Therefore, activation of α1A-ARs via descending LC-NAergic signals may be a common mechanism underlying astrocytic Ca2+ responses in the SDH evoked by noxious stimuli, including chemical irritants.

It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α1A-adrenaline receptors (α1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1aflox/flox mice). A similar reduction was also observed in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α1A-AR antagonist, and by conditional knockout of α1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1aflox/flox mice. Our findings suggest that activation of α1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat.

Pain transmission in the spinal dorsal horn (SDH) is regulated by descending neuronal pathways from the brain, such as noradrenergic (NAergic) neurons from the locus coeruleus. While it is known that spinal NA produces an antinociceptive effect, we have recently shown that NA has an ability to produce pain hypersensitivity via Hes5expressing SDH astrocytes. However, the mechanism underlying the bidirectional effect of spinal NA remains unknown. In this study, we showed that while intrathecal injection of NA at a low dose (NA low ) induced pain hypersensitivity via α 1A -adrenergic receptors (α 1A -ARs) in Hes5 + astrocytes, the hypersensitivity was not observed by intrathecal high-dose NA (NA high ). The effect of NA high was also mediated by activation of inhibitory interneurons via α 1A -ARs. We found that NA high also activated β 1 -ARs in astrocytes that suppressed the astrocytic α 1A -ARs-mediated effect. However, if α 1A -ARs are expressed in inhibitory interneurons, why does NA low produce pain hypersensitivity?We further found that activation of astrocytic α 1A -ARs increased release of adenosine, a factor that suppresses inhibitory interneurons. Therefore, our findings indicate that NA bidirectionally modulates pain transmission via astrocyte-neuron interactions in a concentration-dependent manner.

Astrocytes have become increasingly recognized as critical elements for regulating neuronal development and function in the central nervous system. Recently, we have identified a population of astrocytes (genetically defined by the transcription factor Hes5) in the spinal dorsal horn (SDH) and shown that these astrocytes respond to intraplantar injection of capsaicin (a noxious stimulus) via α 1A -adrenoceptors (α 1A -ARs) activated by locus coeruleus descending noradrenergic (LC-NAergic) signals (Nat. Neurosci., 2020). In this study, to determine whether the activation of SDH astrocytes through α 1A -AR-mediated descending LC-NAergic signals is commonly observed by other noxious stimuli, we examined astrocytic Ca 2+ responses in the SDH after intraplantar injection of formalin using two-photon microscopy. We found that intraplantar formalin induced Ca 2+ responses in SDH astrocytes and that the astrocytic Ca 2+ responses were suppressed by intrathecal pretreatment with an α 1A -AR antagonist. Furthermore, mice lacking α 1A -ARs in Hes5 + astrocytes failed to induce astrocytic Ca 2+ responses in the SDH. Therefore, our findings suggest that activation of SDH astrocytes by noxious stimuli is commonly mediated by activation of α 1A -AR through descending LC-NAergic signals.